Contains basic of pharmaceutical microbiology and major microflora in the cleanroom. Microorganisms like bacteria and fungi. Common microorganisms in the cleanroom and diseases they produce. Biofilm in the pharmaceutical cleanroom.

1. Introduction to
Basic Pharmaceutical Microbiology
Chittaranjan Das

2. MICROBIOLOGY
“Micro” means very small and “biology” is the study of
living things, so microbiology is the study of very small
living organisms such as bacteria, virus, protozoa, fungi
and similar organisms that can't be seen with the naked
eye.

3. Contents
1. Definition and subjects of Pharmaceutical Microbiology
2. Importance of Pharmaceutical Microbiology
3. Microbial contaminations in pharmaceutical
4. Major cleanroom microflora and diseases
Pharmaceutical Microbiology

4. Pharmaceutical Microbiology
Pharmaceutical microbiology is an applied branch of
microbiology that is responsible for ensuring that medications
do not contain harmful levels of microbes- such as bacteria,
yeast and molds.
It mainly focuses on the manufacturing facilities,
manufacturing techniques, process controls and finished
product attributes that limit the harmful effects of
microorganisms on the drug product.

5. Pharmaceutical Microbiology
Pharmaceutical microbiology includes a wide range of subjects,
including:
-microbial identification -aseptic technique & microbial testing
-cleanroom design -environmental monitoring
-contamination control -personnel and material flow
-cleanroom gowning -personnel hygiene & behavior
-cleaning, sanitization, -data trending
and disinfection

6. Importance of Pharmaceutical Microbiology
Pharmaceutical products can save lives and bring back the health
of patients, but what if these products are contaminated?

7. Factors Affecting Microbial Spoilage of Pharmaceuticals

8. Sources of Microbial Contamination in Pharmaceutical

9. Personnel
People are the largest single source of microbial contamination
to the cleanroom environment. Approximately 80% of the
contamination found in a clean room is generated by people.
Human create contaminations that are bacterial, fungal,
chemical, and particulate in nature.

10. Main Source of microflora in Cleanroom
In the nose and ear, pathogenic cocci have been found in 75% of
the subjects examined.
Saliva contains approximately 100 million organisms per milliliter.
Generally, the intestinal tract contains anaerobic, nonsporulating
rods and gram-positive lactobacilli. Some aerobic organisms are
present, but in smaller numbers. They include coliforms, Proteus,
enterococci, and staphylococci.
Hands Skin Respirator Tract Intestinal Tract Ear

11. Cleanroom Microflora

12. Main Microflora in Cleanroom
O Bacteria
O Fungi

13. Bacteria
Bacteria are a type of biological cell. They constitute a large
domain of prokaryotic microorganisms. The bacterial cell is
surrounded by a cell membrane, which is made primarily
of phospholipids. Around the outside of the cell membrane is
the cell wall. Bacterial cell walls are made of peptidoglycan.

14. BACTERIA
Bacteria are subdivided into two classes:
gram-negative organisms and gram-positive organisms.
The former are usually bacilli and are rarely derived from
human sources.
The latter are usually cocci, are frequently found in the air
and are usually derived from a human source and are the
causes of more concerns in cleanroom.

15. Size and Shape of Bacteria
Bacteria vary in size from a cell as small as 0.1–0.2µm in
diameter to those that are >5µm in diameter.
the majority of bacteria are 1–5µm long and 1–2µm in
diameter.
The majority of bacteria are unicellular and possess
simple shapes, e.g. round (cocci), cylindrical (rod) or
ovoid.

16. Bacterial Growth
Bacteria can grow in a multiplicity of environments
ranging from hot sulphur springs (65°C) to deep freezers
(–20°C), from high (pH 1) to low (pH 13) acidity and high
(0.7 M) to low osmolarity (water). Indeed, there is no
natural environment that is free from bacteria.

17. Bacterial Growth
The majority of bacterial cells multiply in number
by a process of binary fission. That is, each individual
will increase in size until it is large enough to divide
into two identical daughter cells.

18. Biofilms
In many natural, medical and industrial settings bacteria
attach to surfaces and form multilayered communities
called biofilms. These commonly contain more than one
species of bacteria, which cooperatively exist together as
a functional, dynamic consortium.

19. Biofilms
Bacterial biofilms are less susceptible to antimicrobial
agents than their free-living, planktonic counterparts.
As a consequence, bacterial biofilms that form on
contaminated medical implants and prosthetic devices,
manufacturing surfaces or fluid conduit systems are
impossible to eliminate with antibiotics or biocides.

20. Pathogenic microorganism
Only a few species of bacteria are disease-producing and are
called pathogenic.
The greater concern are those organisms that, if presented with
the correct set of conditions, can cause disease are called
opportunist pathogens.
Placing the carrier of these organisms (infected or sneezing
person) in a clean room can greatly increase the level of
contamination, thereby increasing the possibility of product
contamination.

21. Microbes present in the Nasal Passageway
Some examples of non-pathogenic bacteria are Streptococcus,
Neisseria, Haemophilus, and Micrococcus.
Some of the pathogenic examples include Staphylococcus
aureus, Corynebacterium diphtheriae, Streptococcus
pneumoniae, and Haemophilus influenzae among others.

22. Microbes present on the hands
In 1938, it is established that bacteria recovered from the hands
could be divided into two categories, namely resident or transient.
The resident flora consists of microorganisms residing under the
superficial cells of the stratum corneum and can also be found on the
surface of the skin. e.g. Staphylococcus epidermidis , corynebacteria,
dermobacteria, and micrococci.
The resident flora is less likely to be associated with infections.

23. Microbes present on the hands
Transient flora (transient microbiota) colonizes the superficial layers of the skin, is
more amenable to removal by routine hand hygiene. Transient microorganisms
do not usually multiply on the skin, but they survive and sporadically multiply on
skin surface.
The transmissibility of transient flora depends on the species present, the
number of microorganisms on the hand surface, and the skin moisture. The
hands of some people may become persistently colonized by pathogenic flora
such as S. aureus, Gram-negative bacilli, or yeast (candida).
Total bacterial counts on the hands of HCWs have ranged from 3.9 × 104 to 4.6 ×
106 CFU/cm2 (39000 – 4,60,00000 CFU/cm2 )

24. Microorganisms on mobile phone
Streptococcus Staphylococcus
Corynebacterium Cold and Flu virus E. Coli
In one study, researchers at the University of Arizona discovered that phones
carry 10 times more bacteria than toilet seats.

25. Micrococcus
Gram-stain positive, non-motile, non-spore-forming spherical
cells. Micrococci have been isolated from human skin. They are
found in many other places in the environment, including water,
dust, and soil. Micrococci can grow well in environments with
little water or high salt concentrations including sportswear
made with synthetic fabrics. Micrococcus are generally non
pathogenic.

26. Micrococcus luteus
Micrococcus luteus is among the most commonly found
organisms in pharmaceutical environmental monitoring.
Micrococcus luteus is Gram positive, spherical, bacterium that
also resides in the nose as well as the mouth, skin (particularly
from the head, arms and legs) and upper resipratory tract as an
obligate aerobe.
Inadequate aseptic techniques and gowning practices, and poor
cleanroom management are frequent sources of M. luteus
contamination.

27. Staphylococcus
This organism may be carried in the nose or throat, on the skin, or
in the hair. Approximately one out of every six people carries S.
aureous.
The optimum pH for the Staphylococcus to live in is 7.0-7.5, and the
optimum temperature is 30-37 degree Celsius.

28. Staphylococcus epidermidis and Staphylococcus aureus
Staphylococcus epidermidis with Corynebacteria predominantly colonizes the
upper respiratory tract, especially the nostrils.
It may form biofilms to attach securely to the epithelial cells in the nasal
passageway. This biofilm, mainly composed of teichoic acid and typically found
on the cell wall of these microbes can also protect them against antibiotics
making them very difficult to treat.
Another Gram-positive bacterium that lives in the human nose is the
Staphylococcus aureus. It is carried either on the skin or in the nose of healthy
people, but 25% - 30% of the population is populated in the nose.

29. Staphylococcal Infection
The Staphylococcus epidermidis and Staphylococcus aureus are
very commonly found gram-positive bacteria which may cause
diseases of the skin and mucous membranes e.g. keratitis and
acne. Normally Staph will not cause any infection, until the
bacteria can enter a wound.
The organisms are frequently detected in saliva and dental plaque
and are thought to be associated with periodontitis.

30. Streptococcus
The Streptococcus pneumoniae is a Gram-positive bacteria that is
commonly found in the mouth, respiratory tract and nasal
passages.
Most Streptococcus cause many diseases e.g. sinusitis ,
pneumonia, blood infections etc.

31. Bacillus
The genus Bacillus originally included all rod-shaped bacteria
comprises only large, spore-forming, Gram-positive bacilli that
form chains and usually grow aerobically or anaerobically.
Under certain conditions the bacteria can produce endospores
that enable them to survive in inhospitable environments ,
temperature range from 5°C to 50°C, with an optimum growth of
between 28°C to 35°C, pH range 5 to 9. They are common
environmental organisms, frequently isolated in laboratories as
contaminants of media or specimens.

32. Bacillus cereus
The most common microorganism associated with contamination
in pharmaceutical is Bacillus cereus. The main reason for the
detection of species of Bacillus in pharmaceutical products,
according to FDA warning letters, relates to inadequate cleaning
and disinfection system. This includes a failure to include a
sporicidal agent as part of the disinfection system or, failure to
qualify a sporicide used to kill or inactivate spores. A media fill
failure had occurred and been linked to a Bacillus contaminant.
B. cereus is also known to cause chronic skin infections (keratitis).

33. Bacillus oleronius
Bacillus oleronius causes rosacea skin infection.

34. Pseudomonas aeruginosa
Pseudomonas aeruginosa is a common encapsulated, Gram-
negative, rod-shaped bacterium that can cause disease in plants and
animals, including humans. Bacterial keratitis is a bacterial infection
of the cornea can follow from an injury or from wearing contact
lenses. The bacteria involved for contact lens wearers
is Pseudomonas aeruginosa

35. Corynebacterium
Corynebacterium is a Gram-positive normal flora found in the
human nose.
Most species of Corynebacterium will not cause diseases in
humans; however, there is a specific species that is highly
infectious; the Corynebacterium diphtheriae causes infection in
the upper respiratory tract, and can be deadly if untreated.

36. E. Coli
Escherichia coli, also known as E. coli, is a Gram-negative,
facultative anaerobic, rod-shaped, coliform bacterium.

37. E. Coli Infection
Most E. coli strains are harmless, but pathogenic varieties
cause serious food poisoning, septic shock, meningitis, or
urinary tract infections in humans.

38. Salmonella
Salmonella is a genus of rod-shaped (bacillus) Gram-negative
bacteria of the family Enterobacteriaceae. The common
species of Salmonella is Salmonella enterica.

39. Salmonella Infection
Salmonella enterica causes infection and the most common
symptoms are diarrhea, fever, abdominal cramps, and vomiting.
Typhoid Fever, also known simply as typhoid, is a Salmonella
infection due to a specific type of Salmonella bacteria.

40. Fungi
The kingdom of organisms that essentially includes yeast,
molds, and mushrooms, is termed as fungi.
all the body’s normal flora categorically contains several
fungi. However, most fungi are not pathogenic in nature.
In immunocompetent humans these fungi usually cause
minor infections of the hair, nails, mucous membranes, or
skin.

41. Fungi in Pharmaceutical Clean Room
The two groups of fungi that have practical importance in
the cleanroom are molds and yeasts. Fungal spores range
in sizes from 1–50 µm and are easily dispersed into
the cleanroom environment through many routes.

42. Mold and Yeast
A mold is a fungus that grows in the form of multicellular
filaments called hyphae.
In contrast, Yeast is a single cell organism, which is usually a
thread and filamentous in shape.

43. Aspergillus niger
The causative agent of ‘black mould’ on the outside of onions.
Highly resistant to 70% IPA (2 log reduction on exposure to 2
minutes) and can be found on the various surfaces of cleanroom.
A. niger is one of the most common causes of otomycosis (fungal
ear infections), which can cause pain, temporary hearing loss.
Colonies of Aspergillus niger on slant
Sabourard's agar in the test tubes.

44. Aspergillus fumigatus
A. fumigatus occurs in areas with widely different climates and
environments. Aspergillus fumigatus can grown on certain
building materials. It is less resistance to 70% IPA (6 log
reduction on exposure of 2 minutes).
This fungus is capable to grow at 37 °C or 99 °F (normal human
body temperature). A. fumigatus can cause chronic pulmonary
infections, allergic bronchopulmonary aspergillosis, or allergic
disease in immunocompetent hosts.

45. Candida albicans
Candida albicans is an opportunistic pathogenic yeast that is a
common member of the human gut flora. It can also survive
outside the human body. It is detected in the gastrointestinal
tract and mouth in 40–60% of healthy adults.

46. Candida Infections (candidiasis)
In cutaneous candidiasis, the skin is infected with candida fungi.
This type of infection is fairly common. It can involve almost any
skin on the body, but most often it occurs in warm, moist, creased
areas such as the armpits and groin.

47. References
1. Wikipedia
2. www.steris.com
3. ResearchGate: A Review of Cleanroom Microflora: Types, Trends, and patterns
4. Journal.pda.org
5. EPM Magazine
6. www.cordenpharma.com
7. Google images
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